European Journal of Pharmaceutics and Biopharmaceutics (v.77, #2)

APV Diary (I).

Manufacture and characterization of mucoadhesive buccal films by Javier O. Morales; Jason T. McConville (187-199).
In dry dosage forms in contact with surfaces with a thin mucus layer, such as a buccal mucoadhesive film, two stages need to occur to establish the mucoadhesive bond, namely a contact and a consolidation stage.The buccal route of administration has a number of advantages including bypassing the gastrointestinal tract and the hepatic first pass effect. Mucoadhesive films are retentive dosage forms and release drug directly into a biological substrate. Furthermore, films have improved patient compliance due to their small size and reduced thickness, compared for example to lozenges and tablets. The development of mucoadhesive buccal films has increased dramatically over the past decade because it is a promising delivery alternative to various therapeutic classes including peptides, vaccines, and nanoparticles. The “film casting process” involves casting of aqueous solutions and/or organic solvents to yield films suitable for this administration route. Over the last decade, hot-melt extrusion has been explored as an alternative manufacturing process and has yielded promising results. Characterization of critical properties such as the mucoadhesive strength, drug content uniformity, and permeation rate represent the major research areas in the design of buccal films. This review will consider the literature that describes the manufacture and characterization of mucoadhesive buccal films.
Keywords: Buccal drug delivery; Oral mucosa; Mucoadhesion; Permeation; Mucoadhesive polymers; Buccal patches;

Reversible Targeting and controlled release delivery of daunorubicin to cancer cells by aptamer-wrapped carbon nanotubes by Seyed Mohammad Taghdisi; Parirokh Lavaee; Mohammad Ramezani; Khalil Abnous (200-206).
Effects of Dau and Dau-aptamer-SWNTs tertiary complex on control and target cells viability (MTT assay). Cells were treated with aptamer-SWNTs, Dau, and Dau-aptamer-SWNTs tertiary complex for 3 h. After 72 h post-treatment viability of the cells was assessed using MTT technique. * Indicates there is a significant difference between viability of Molt-4 and U266 cells after treatment with Dau-aptamer-SWNTs tertiary complex (p  < 0.005).Single-walled carbon nanotubes (SWNTs) have been already used as drug carriers. In this study, we introduced sgc8c aptamer (this aptamer targets leukemia biomarker protein tyrosine kinase-7) to complex between Dau (daunorubicin) and SWNT to enhance targeted delivery of Dau to acute lymphoblastic leukemia T-cells (Molt-4).Dau-aptamer-SWNTs tertiary complex formation was analyzed by visible spectroscopy and spectrofluorophotometric analysis. Dau release profiles from the complex were investigated in pH 7.4 and 5.5. For cytotoxic studies (MTT assay), Molt-4 (target) and U266 (B lymphocyte human myeloma, non-target) cells were treated with Dau, Dau-aptamer-SWNTs tertiary complex. Internalization was analyzed by flow cytometry. Targeted delivery of Dau was antagonized using antisense of aptamer.Dau was efficiently loaded onto SWNTs (efficiency ∼157%). Dau was released from Dau-aptamer-SWNTs tertiary complex in a pH-dependent manner (higher release rate at pH 5.5). Flow cytometric analysis showed that the tertiary complex was internalized effectively to Molt-4 cells, but not to U266 cells. Cytotoxicity of Dau-aptamer-SWNTs tertiary complex also confirmed internalization data. Dau-aptamer-SWNTs tertiary complex was less cytotoxic in U266 cells when compared to Dau alone. No significant change in viability between Dau- and complex-treated Molt-4 cells was observed. Cytotoxicity of Dau-aptamer-SWNTs complex was efficiently and quickly reversed using antisense in Molt-4 cells.Dau-aptamer-SWNTs complex is able to selectively target Molt-4 cells. The other advantages of this system are reversibility and pH-dependant release of Dau from its complex.
Keywords: Aptamer; SWNTs; Leukemia; Daunorubicin; Targeted delivery; Cytotoxicity;

Preclinical evaluation of tacrolimus colloidal dispersion for inhalation by Alan B. Watts; Jay I. Peters; Robert L. Talbert; Kevin P. O’Donnell; Jacqueline J. Coalson; Robert O. Williams (207-215).
Histological staining of lungs with (a) H&E, (b) CD 68, and (c) PAS for the evaluation of tacrolimus-induced cellular inflammation, monocyte/macrophage presence, and mucus production, respectively, in rodent lungs after 28 consecutive days dosing.Substantial improvements in transplant therapy have been made in the past four decades resulting in the acceptance of organ transplantation as a viable treatment for late-stage disease and organ failure. More recently, lung transplantation has gained acceptance; however, high incidence of chronic rejection and opportunistic infections has limited success rates in comparison with other transplant procedures. To achieve more targeted therapy, pulmonary administration of nebulized tacrolimus (TAC) colloidal dispersion once daily for 28 consecutive days in Sprague Dawley (SD) rats has been investigated for safety and systemic elimination. A liquid dispersion of colloidal TAC and lactose (1:1 ratio by weight) was aerosolized using a vibrating mesh nebulizer and administered via a nose-only dosing chamber. Blood chemistry and histological comparisons to saline-dosed animals showed no clinically significant differences in liver and kidney function or lung tissue damage. Maximum blood and lung concentrations sampled 1 h after the final dose showed TAC concentrations of 10.1 ± 1.4 ng/mL and 1758.7 ± 80.0 ng/g, respectively. Twenty-four hours after the final dose, systemic TAC concentrations measured 1.0 ± 0.5 ng/mL, which is well below clinically accepted trough concentrations (5–15 ng/mL) for maintenance therapy, and therefore, would not be expected to induce toxic side effects. The propensity for pulmonary retention seen when compared to single dose lung levels may be due to macrophage uptake and the lipophilic nature of TAC. Additionally, three month stability testing of TAC powder for reconstitution showed no changes in amorphous nature or drug potency when stored at ambient conditions. TAC colloidal dispersion proved to be non-toxic when administered by pulmonary inhalation to SD rats over 28 days while providing therapeutic concentrations locally. This delivery strategy may prove safe and effective for the prevention of lung allograft rejection in lung transplant recipients.
Keywords: Tacrolimus; Inhalation; Preclinical; Colloidal; Amorphous; Safety;

In vitro and in vivo evaluation of WGA–carbopol modified liposomes as carriers for oral peptide delivery by Abdallah Makhlof; Shiho Fujimoto; Yuichi Tozuka; Hirofumi Takeuchi (216-224).
Simultaneous tissue adhesion and penetration of WGA and liposomes through the small intestine of rats after oral administration of WGA–CP liposomes.Surface modification of liposomal nanocarriers with a novel polymer–lectin conjugate was proposed for enhancing the systemic uptake of encapsulated peptide and protein therapeutics after oral administration. Wheat germ agglutinin (WGA) was covalently attached to carbopol (CP) using the carbodiimide method. The prepared WGA–CP conjugate retained the biological cell binding activity of WGA without any evidence of cytotoxicity to Caco-2 monolayers. Cationic liposomes in the size range of 100 nm were prepared by the lipid film hydration method followed by probe sonication and surface modification with negatively charged WGA–CP. The uptake of WGA–CP liposomes by Caco-2 cells was significantly higher than that of non-modified or CP liposomes. The uptake was dependent on the surface concentration of WGA, temperature, and incubation period and was significantly inhibited in the presence of chlorpromazine and 10-fold excess of free WGA. These results suggest the involvement of active transport mechanism for the cellular uptake of the modified liposomes, mediated mainly by binding of WGA to its specific cell membrane receptors. Dual channel confocal microscopy confirmed the simultaneous association and internalization of the polymer conjugate and the liposomal carrier by Caco-2 cells and intestinal membrane of rats. In addition, the pharmacological efficacy of calcitonin, a model peptide drug, was enhanced by more than 20- and 3-fold following peroral administration of calcitonin-loaded WGA–CP liposomes when compared to non-modified and CP liposomes, respectively.
Keywords: Lectins; Wheat germ agglutinin; Bioadhesion; Liposomes; Oral peptide delivery; Calcitonin;

Thermosensitive hydrogels for nasal drug delivery: The formulation and characterisation of systems based on N-trimethyl chitosan chloride by H. Nazar; D.G. Fatouros; S.M. van der Merwe; N. Bouropoulos; G. Avgouropoulos; J. Tsibouklis; M. Roldo (225-232).
Towards the development of a thermosensitive drug-delivery vehicle for nasal delivery, a systematic series of N-trimethyl chitosan chloride polymers, synthesised from chitosans of three different average molecular weights, have been co-formulated into a hydrogel with poly(ethylene glycol) and glycerophosphate. Rheological evaluations have shown that hydrogels derived from N-trimethyl chitosan with a low degree of quaternisation and high or medium average molecular weight exhibit relatively short sol-gel transition times at physiologically relevant gelation temperatures. Also, the same hydrogels display good water-holding capacity and strong mucoadhesive potential, and their mixtures with mucus exhibit rheological synergy. An aqueous hydrogel formulation, derived from N-trimethyl chitosan of medium average molecular weight and low degree of quaternisation, appears particularly promising in that it exhibits most favourable rheological and mucoadhesive behaviour and a sol-gel transition that occurs at 32.5 °C within 7 min.Towards the development of a thermosensitive drug-delivery vehicle for nasal delivery, a systematic series of N-trimethyl chitosan chloride polymers, synthesised from chitosans of three different average molecular weights, have been co-formulated into a hydrogel with poly(ethylene glycol) and glycerophosphate. Rheological evaluations have shown that hydrogels derived from N-trimethyl chitosan with a low degree of quaternisation and high or medium average molecular weight exhibit relatively short sol–gel transition times at physiologically relevant temperatures. Also, the same hydrogels display good water-holding capacity and strong mucoadhesive potential, and their mixtures with mucus exhibit rheological synergy. An aqueous hydrogel formulation, derived from N-trimethyl chitosan of medium average molecular weight and low degree of quaternisation, appears particularly promising in that it exhibits most favourable rheological and mucoadhesive behaviour and a sol–gel transition that occurs at 32.5 °C within 7 min.
Keywords: Chitosan; N-trimethyl chitosan chloride; Thermosensitive; Mucoadhesion; Hydrogel; Nasal drug delivery;

The design of flexible ciprofloxacin-loaded PLGA implants using a reversed phase separation/coacervation method by Peter In Pyo Park; Michael Makoid; Sriramakamal Jonnalagadda (233-239).
This research shows that flexible implants can be designed from PLA and PLGA polymers using a reversed phase separation/coacervation process. The resulting implants show significantly lower glass transition values, have a flexible characteristics, and can sustain the release of ciprofloxacin HCl for several days in vitro.The purpose of this research is to design and characterize flexible PLGA-based implants for the controlled release of ciprofloxacin hydrochloride for up to 6 weeks in vitro. This research uses a reversed phase separation/coacervation method to fabricate flexible PLA and PLGA: excipient implants with dichloromethane/mineral oil as solvent/non-solvent. Physical characterization was performed using thermal and mechanical analyses. Drug loading and release studies were performed with ciprofloxacin HCl as the model drug. Release kinetics was modeled to elucidate possible mechanisms of drug release. Four polymer–excipient combinations with glass transition temperatures less than 20 °C and representing a wide range of Young’s moduli were shown to entrap up to 8% of ciprofloxacin HCl that could be released at a controlled rate for 65 days in vitro. The release rate could consistently fit a ternary Gaussian pattern with an R 2  > 0.99. It was postulated that these release patterns could be related to ciprofloxacin that was loosely or poorly bound (burst release), trapped within the polymer matrix, or encapsulated by the polymer. These studies show that flexible implants can be fabricated from PLGA-based polymers for the controlled release of ciprofloxacin hydrochloride for up to 6 weeks in vitro.
Keywords: Flexible; Biodegradable; Ciprofloxacin; Coacervation; PLGA;

Adsorbents in transdermal matrix systems.The drug release from medium molecular weight polyisobutene patches containing adsorbates (drug content: 0.2% ethinyl estradiol, 1.0% levonorgestrel; adsorbent content: 20%, w/w) increased in the order of no adsorbent < titanium dioxide < MCC < crospovidone. This was attributed to differences in drug crystallinity which increased in the order of crospovidone (crystal free) < MCC < titanium dioxide < no adsorbent and the water uptake which increased in the order of no adsorbent (0.1%) = titanium dioxide (0.1%) < MCC (1.6%) < crospovidone (4.8%) at 90% rh. Patches containing adsorbates onto crospovidone were investigated in detail. Increasing the adsorbate’s drug loading increased the drug release up to a crospovidone content of 15% (w/w). Patches were crystal free for crospovidone contents ⩾10% (w/w), which corresponds to a drug loading of crospovidone of 12% (w/w). In conclusion, the incorporation of drug adsorbates onto crospovidone into patches based on polyisobutene significantly increased the drug release (approximately 9.1 times for ethinyl estradiol and 15.4 times for levonorgestrel) and prevented drug recrystallization.
Keywords: Transdermal drug delivery; Absorbent; Crospovidone; Ethinyl estradiol; Levonorgestrel; Drug recrystallization;

The influence of positive or negative charges in the passive and iontophoretic skin penetration of porphyrins used in photodynamic therapy by Guilherme M. Gelfuso; Taís Gratieri; Joel G. Souza; José A. Thomazine; Renata F.V. Lopez (249-256).
Fluorescence microscopy photomicrographs of vertical slicing of rat skin. The red color illustrates drugs’ skin permeation. Iontophoresis enhanced the delivery of the negative drug, TPPS4, only to the superficial layers of rat skin, while structurally similar but positive TMPyP was transported in higher amounts to deeper skin with homogeneous distribution when the electric current was applied. Meso-tetra-(N-methylpiridinium-4-yl)-porphyrin (TMPyP) and meso -tetra-(4-sulfonatophenyl)-porphyrin (TPPS4) are photosensitizing drugs (PS) used in photodynamic therapy (PDT). Based on the fact that these compounds present similar chemical structures but opposite charges at pH levels near physiological conditions, this work aims to evaluate the in vitro and in vivo influence of these electrical charges on the iontophoretic delivery of TMPyP and TPPS4, attempting to achieve maximum accumulation of PS in skin tissue. The iontophoretic transport of these drugs from a hydrophilic gel was investigated in vitro using porcine ear skin and vertical, flow-through diffusion cells. In vivo experiments using rats were also carried out, and the penetration of the PSs was analyzed by fluorescence microscopy to visualize the manner of how these compounds were distributed in the skin after a short period of iontophoresis application. In vitro, both passive and iontophoretic delivery of the positively charged TMPyP were much greater (20-fold and 67-fold, respectively) than those of the negatively charged TPPS4. TPPS4 iontophoresis in vivo increased the fluorescence of the skin only in the very superficial layers. On the other hand, iontophoresis of the positively charged drug expressively increased the rat epidermis and dermis fluorescence, indicating high amounts of this drug throughout the skin layers. Moreover, TMPyP was homogeneously distributed around and into the nuclei of the skin cells, suggesting its potential use in topical PDT.
Keywords: Iontophoresis; Electrical charge; Porphyrins; Topical delivery; Photodynamic therapy;

Biorelevant in vitro dissolution testing of products containing micronized or nanosized fenofibrate with a view to predicting plasma profiles by Daniel Juenemann; Ekarat Jantratid; Christian Wagner; Christos Reppas; Maria Vertzoni; Jennifer B. Dressman (257-264).
The graphical abstract shows: the difference in dissolution profile when using an appropriate and an inappropriate filter pore size (above), the resulting plasma profile if bioavailability is determined by dissolution rate for both the nanosized and microsized formulation (middle), the resulting plasma profile if bioavailability is limited by absorption for the nanosized formulation.The ability of in vitro biorelevant dissolution tests to predict the in vivo performance of nanosized fenofibrate (Lipidil 145 ONE®) and microsized fenofibrate (Lipidil – Ter®) was evaluated in this study. In vitro dissolution was carried out using USP apparatus 2 (paddle method) with updated biorelevant media to simulate the pre- and postprandial states. Membrane filters with different pore sizes were evaluated for their ability to hold back undissolved, nanosized drug particles. It was shown that filters with pore sizes of 0.1 μm and 0.02 μm were able to separate molecularly dissolved drug from colloidal and undissolved particles. In vitro results obtained with a suitable filter were used to generate simulated plasma profiles in combination with two different models using STELLA® software: (a) under the assumption of no permeability restrictions to absorption and (b) under the assumption of a permeability restriction. The simulated plasma profiles were compared to in vivo data for the nanosized and the microsized formulation in the fasted and fed states. The first model approach resulted in good correlation for the microsized fenofibrate formulation, but the plasma profile of the formulation containing nanosized fenofibrate was overpredicted in the fasted state. The second model successfully correlated with in vivo data for both formulations, regardless of prandial state. Comparison of simulations with the two models indicates that in the fasted state, absorption of fenofibrate from the nanosized formulation is at least partly permeability-limited, while for the microsized formulation the dissolution of fenofibrate appears to be rate-determining.
Keywords: Biorelevant dissolution testing; In vitro - in vivo correlations (IVIVC); Fenofibrate; Nanosized; Filter pore sizes; Food effect;

Uptake and permeability studies of BBB-targeting immunoliposomes using the hCMEC/D3 cell line by Eleni Markoutsa; Georgios Pampalakis; Anna Niarakis; Ignacio A. Romero; Babette Weksler; Pierre-Olivier Couraud; Sophia G. Antimisiaris (265-274).
Confocal microscopy of hCMEC/D3 monolayers formed on transwell membranes. Cells were treated with control liposomes (control), murine serum IgG-immunoliposomes (IgG), and OX-26 immunoliposomes (OX-26). All liposomes are stained with lipid–rhodamine (red) and encapsulate FITC (green). Nuclei are stained with DAPI (blue). Each column represents different slices obtained under the confocal microscope (3.75 μm/slice).The targeting potential of OX-26-decorated immunoliposomes was investigated, using the human brain endothelial cell line hCMEC/D3 as a model of the blood–brain barrier (BBB). Immuno-nanoliposomes were prepared by the biotin/streptavidin ligation strategy, and their uptake by hCMEC/D3 cells and permeability through cell monolayers was studied. In order to elucidate the mechanisms of uptake, pH-sensitive fluorescence signal of HPTS was used, while transport was measured using double labeled immunoliposomes (with aqueous and lipid membrane fluorescent tags). PEGylated and non-specific-IgG-decorated liposomes were studied under identical conditions, as controls. CHO-K1 cells (which do not overexpress the transferrin receptor) were studied in some cases for comparative purposes.Experimental results reveal that hCMEC/D3 cells are good models for in vitro screening of BBB-targeting nanoparticulate drug delivery systems. Uptake and transcytosis of immunoliposome-associated dyes by cell monolayers was substantially higher compared to those of control liposomes. HPTS-entrapping OX-26-immunoliposome uptake indicated lysosomal localization and receptor-mediated mechanism. The ratio of aqueous/lipid label transport is affected by pre-incubation with antibody, or use of high lipid doses, suggesting that vesicles are transported intact after lysosome saturation. Co-decoration with a second ligand slightly decreases OX-26-decorated vesicle uptake, but not transcytosis, proving that the biotin–streptavidin technique can be applied for the generation of dual-targeting nanoliposomes.
Keywords: Dual targeting; Blood–brain barrier; Transcytosis; Immunoliposomes; Transferrin; OX-26 antibody;

The events related to transport of curcumin across Caco-2 cell line monolayer confirmed absence of P-gP efflux and active transport. Permeability was adversely affected by chemical degradation and intracellular accumulation.Curcumin a poly-phenolic compound possesses diverse pharmacologic activities; however, its development as a drug has been severely impeded by extremely poor oral bioavailability. Poor aqueous solubility and extensive metabolism have been implicated for this but the role of membrane permeability has not been investigated. In the present study, permeability of curcumin was assessed using the Caco-2 cell line. Curcumin was poorly permeable with a P app (A → B) value of 2.93 ± 0.94 × 10−6  cm/s. P app value in (B → A) study was found out to be 2.55 ± 0.02 × 10−6  cm/s, thus ruling out the role of efflux pathways in poor oral bioavailability of curcumin. Studies using verapamil, a P-gp inhibitor, further confirmed this finding. Detailed mass balance studies showed loss of curcumin during transport. Further experiments using lysed cells revealed that 11.78% of curcumin was metabolized during transport. Studies using itraconazole, a CYP3A4 inhibitor, established its role in curcumin metabolism. Curcumin was also found to accumulate in cells as revealed by CLSM studies. Sorption and desorption kinetic studies further confirmed accumulation of curcumin inside the cells. Amount accumulated was quantitated by HPLC and found to be >20%. Thus, intestinal first-pass metabolism and intracellular accumulation played a role in poor permeability of curcumin. Based on its poor aqueous solubility and intestinal permeability, curcumin can be classified as a BCS Class IV molecule. This information can facilitate designing of drug delivery systems for enhancement of oral bioavailability of curcumin.
Keywords: Curcumin; Caco-2 cells; Efflux; Permeability; Metabolism; Accumulation;

The production of ‘aerodynamically equivalent’ drug and excipient inhalable powders using a novel fractionation technique by Mohammed Taki; Christopher Marriott; Xian-Ming Zeng; Gary P. Martin (283-296).
The aerodynamic fractionation technique uniquely enables the pre-determination of particle size distribution of inhalation powders and can be equally applied to various drugs and excipients regardless of their previous production/processing history. Fractionation may also be used to compare and contrast samples produced by different processes.Inhalation particles can be produced by various techniques such as milling, controlled crystallisation and spray-drying, but current methods cannot, to-date, precisely control the aerodynamic size distribution of produced powders. The aim of this study was to develop and validate a novel preparative technique whereby the efficient and reproducible aerodynamic fractionation of drug and excipient powders could be achieved. Salmeterol xinafoate (SX), fluticasone propionate (FP) and fine α-lactose monohydrate (FL) were chosen as model compounds. Powders were aerosolised using a dry powder feeder into a Next Generation Impactor operated at 60 L min−1. Powders deposited on NGI stages were then collected and analysed. The fractionation process was successful for all powders producing significant linear correlations between the pre-set aerodynamic cut-off limits and geometric size measurements. For each of SX, FP and FL, sufficient powder quantities were recovered from NGI stages 1–6 producing six fractions with sequential aerodynamic and geometric particle size distributions. The fractionation technique was efficient and reproducible for all powders studied. The method can be equally applied to various drugs and excipients regardless of their previous production/processing history. Therefore, the aerodynamic fractionation technique may be used to compare and contrast samples produced by different processes.
Keywords: Particle size distribution; Aerodynamic fractionation; Salmeterol xinafoate; Fluticasone propionate; Aerodynamically equivalent powders;

Ethylene vinyl acetate as matrix for oral sustained release dosage forms produced via hot-melt extrusion by A. Almeida; S. Possemiers; M.N. Boone; T. De Beer; T. Quinten; L. Van Hoorebeke; J.P. Remon; C. Vervaet (297-305).
Characterisation and drug release from ethylene-vinyl acetate matrices.Different ethylene vinyl acetate grades (EVA9, EVA15, EVA28 and EVA40 having a VA content of 9%, 15%, 28% and 40%, respectively) were characterized via differential scanning calorimetry. Glass transition temperature (T g), polymer crystallinity, melting point and polymer flexibility were positively influenced by the vinyl acetate content. The processability of EVA-based formulations produced by means of hot-melt extrusion (2 mm die) was evaluated in function of VA content, extrusion temperature (60–140 °C) and metoprolol tartrate (MPT, used as model drug) concentration (10–60%). Matrices containing 50% MPT resulted in smooth-surfaced extrudates, whereas at 60% drug content severe surface defects (shark skinning) were observed. Drug release from EVA/MPT matrices (50/50, w/w) was affected by the EVA grades: 90% after 24 h for EVA15 and 28, while EVA9 and EVA40 formulations released 80% and 60%, respectively. Drug release also depended on drug loading and extrusion temperature. For all systems, the total matrix porosity (measured by X-ray tomography) was decreased after dissolution due to elastic rearrangement of the polymer. However, the largest porosity reduction was observed for EVA40 matrices as partial melting of the structure (melt onset temperature: 34.7 °C) also contributed (thereby reducing the drug release pathway and yielding the lowest release rate from EVA40 formulations).The Simulator of the Human Intestinal Microbial Ecosystem (SHIME) used to evaluate the stability of EVA during gastrointestinal transit showed that EVA was not modified during GI transit, nor did it affect the GI ecosystem following oral administration.
Keywords: Hot-melt extrusion; Ethylene vinyl acetate; Sustained release; Multiple-unit dosage form; Matrix system; Metoprolol tartrate;

Novel extended-release formulation of lovastatin by one-step melt granulation: In vitro and in vivo evaluation by L. Ochoa; M. Igartua; R.M. Hernández; A.R. Gascón; M.A. Solinis; J.L. Pedraz (306-312).
The novel extended-release formulation of lovastatin developed exhibits zero-order release in vitro and provides extended plasma levels of lovastatin and its active metabolite after oral administration to dogs.The objective of this study was to apply a one-step melt granulation method to develop an extended-release formulation of lovastatin (LOV-ER). We prepared a formulation using PEG 6000 as binder agent in a laboratory scale high-shear mixer. In vitro dissolution studies showed that the release of the drug from the new formulation followed a zero-order kinetic with no differences in the release profile with either the pH media or the agitation rate. The pharmacokinetic of lovastatin and its metabolite lovastatin acid was evaluated after the administration of the new formulation to Beagle dogs in fasted conditions and after a high-fat meal, and compared to the marketed formulation Altoprev®. After the administration of LOV-ER, extended plasma profiles of lovastatin and its active metabolite were achieved in both fasted conditions and after the high-fat meal. Plasma levels of lovastatin and lovastatin acid were always higher when the LOV-ER formulation was administered with the high-fat meal. A high variability in plasma levels and pharmacokinetic parameters was obtained, being this variability higher when the formulation was administered under fasting conditions. Our results suggest that there is an increase in lovastatin bioavailability when the formulation is administered after the high-fat meal. When we compare LOV-ER and Altoprev®, both administered after the high-fat meal, we found significant differences (p  < 0.05) in C max of lovastatin and in AUC0–∞ and MRT of lovastatin acid. No differences were detected between both formulations in fasting conditions. In this regard, the high-fat meal seems to increase the absorption extent of lovastatin from LOV-ER formulation and to delay the absorption rate of the drug from Altoprev®. In conclusion, we developed a lovastatin formulation that provided extended plasma levels that confirm that one-step melt granulation in high-shear mixer could be an easy and cost-effective technique for extended-release formulation development.
Keywords: Lovastatin; Beagle dog; Melt granulation; Extended release; High-shear mixer;

Food-dependent disintegration of immediate release fosamprenavir tablets: In vitro evaluation using magnetic resonance imaging and a dynamic gastrointestinal system by Joachim Brouwers; Bart Anneveld; Gert-Jan Goudappel; Guus Duchateau; Pieter Annaert; Patrick Augustijns; Evelijn Zeijdner (313-319).
A postprandial delay in gastric disintegration of immediate release fosamprenavir tablets, causing postponed absorption in humans, is simulated in the dynamic gastrointestinal model TIM-1 and visualized using magnetic resonance imaging.In the present study, we demonstrated the value of two advanced tools, the TNO gastric and small Intestinal Model (TIM-1) and magnetic resonance imaging (MRI), for the in vitro evaluation of food-dependent disintegration of immediate release fosamprenavir tablets. Upon introduction of a tablet with the nutritional drink Scandishake Mix® in the stomach compartment of TIM-1, simulating the fed state, disintegration and fosamprenavir dissolution were significantly postponed compared to the fasted state (lag time 80 ± 23 min). This resulted in a lag in the appearance of bioaccessible fosamprenavir (<5% during the first 2 h), even though the nutritional state did not significantly alter the cumulative bioaccessibility after 5 h. These results were in agreement with the previously observed postprandial delay in gastric fosamprenavir tablet disintegration and subsequent amprenavir absorption in healthy volunteers. Therefore, TIM-1 can be used in tablet development to identify food-induced disintegration issues causing unexpected clinical behavior. From a mechanistic perspective, we applied MRI to illustrate impaired water ingress in fosamprenavir tablets immersed in the nutritional drink compared to simulated gastric fluid. This effect may be attributed to both competition between nutritional components and the tablet for the available water (indicated by reduced rotational and translational diffusion) as well as the possible formation of a food-dependent precipitation layer on the HPMC-coated tablet.
Keywords: Formulation development; Immediate release tablets; Tablet disintegration; Fed state; Magnetic resonance imaging; TNO gastrointestinal model;

Swelling kinetics of spray-dried chitosan acetate assessed by magnetic resonance imaging and their relation to drug release kinetics of chitosan matrix tablets by Kampanart Huanbutta; Pornsak Sriamornsak; Sontaya Limmatvapirat; Manee Luangtana-anan; Yasuo Yoshihashi; Etsuo Yonemochi; Katsuhide Terada; Jurairat Nunthanid (320-326).
A high potential technique, magnetic resonance imaging (MRI), was used to monitor in situ swelling behavior of spray dried chitosan acetate, CSA, in various media i.e, 0.1 N HCl, pH 6.8 and 5.0 Tris HCl buffers. The linear relationship between percentage of swelling and drug release from CSA matrix tablets exhibited the important role of the swelling behavior of the polymer on controlling the drug release.Magnetic resonance imaging (MRI) was used to assess in situ swelling behaviors of spray-dried chitosan acetate (CSA) in 0.1 N HCl, pH 6.8 and pH 5.0 Tris–HCl buffers. The in vitro drug releases from CSA matrix tablets containing the model drugs, diclofenac sodium and theophylline were investigated in all media using USP-4 apparatus. The effect of chitosan molecular weight, especially in pH 6.8 Tris–HCl, was also studied. In 0.1 N HCl, the drug release from the matrix tablets was the lowest in relation to the highest swelling of CSA. The swelling kinetics in Tris–HCl buffers are Fickian diffusion according to their best fit to Higuchi’s model as well as the drug release kinetics in all the media. The high swelling rate ( k s ′ ) was found to delay the drug release rate (k′). The linear relationship between the swelling and fractions of drug release in Tris–HCl buffers was observed, indicating an important role of the swelling on controlling the drug release mechanism. Additionally, CSA of 200 and 800 kDa chitosan did not swell in pH 6.8 Tris–HCl but disintegrated into fractions, and the drug release from the matrix tablets was the highest.
Keywords: Chitosan acetate; Magnetic resonance imaging; Tablet; Swelling;

Affinity and translocation relationships via hPEPT1 of H-Xaa-Ser-OH dipeptides: Evaluation of H-Phe-Ser-OH as a pro-moiety for ibuprofen and benzoic acid prodrugs by Diana Højmark Omkvist; Dennis Jespersen Trangbæk; Jemma Mildon; James S. Paine; Birger Brodin; Mikael Begtrup; Carsten Uhd Nielsen (327-331).
Solid phase synthesis of Xaa-Ser dipeptides.The intestinal di/tri-peptide transporter 1 (hPEPT1) has been suggested as a drug delivery target for peptide-based prodrugs. The aim of the study was to synthesize a series of 11 serine-containing dipeptides (H-Xaa-Ser-OH) and to investigate the relationship between binding to and transport via hPEPT1. An additional aim was to design a dipeptide which could serve as a pro-moiety for prodrugs targeted to hPEPT1. Xaa was chosen from the 20 proteogenic amino acids. The dipeptides were synthesized using solid phase peptide synthesis. The Ki -values of H-Xaa-Ser-OH dipeptides for hPEPT1 in MDCK/hPEPT1 cells ranged from 0.14 mM (log IC50  = −0.85 ± 0.06) for H-Tyr-Ser-OH to 0.89 mM (log IC50  = −0.09 ± 0.02) for H-Gly-Ser-OH, as measured in a competition assay with [14C]Gly-Sar. The dipeptides were translocated via hPEPT1 with Km -values in the range of 0.20 (log IC50  = −0.69 ± 0.04) for H-Met-Ser-OH to 1.04 (log IC50  = 0.02 ± 0.04) mM for H-Gly-Ser-OH. The relationship between ligand and transportate properties indicated that the initial binding of the ligand to hPEPT1 is the major determinant for translocation of the investigated dipeptides. H-Phe-Ser-OH was selected as a pro-moiety, and two prodrugs were synthesized, i.e. H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH. Both H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH had high affinity for hPEPT1 with Ki -values of 0.07 mM (log IC50  = −0.92 ± 0.12) and 0.12 mM (log IC50  = −1.17 ± 0.40), respectively. However, none of the prodrugs were translocated via hPEPT1. This indicated that the coupling of the drug compounds to the peptide backbone did not decrease transporter binding, but abolished translocation, and that high affinity of prodrugs does not necessarily translate into favourable permeation properties.
Keywords: hPEPT1; Dipeptide; Prodrug; H-Xaa-Ser-OH; Affinity; Translocation;

Haloperidol-loaded polysorbate-coated polymeric nanocapsules increase its efficacy in the antipsychotic treatment in rats by Dalila M. Benvegnú; Raquel C.S. Barcelos; Nardeli Boufleur; Patrícia Reckziegel; Camila S. Pase; Aline F. Ourique; Ruy Carlos R. Beck; Marilise E. Bürger (332-336).
Haloperidol-loaded polysorbate-coated nanocapsules (H-Nc) showed nanometric size with high efficiency of encapsulation (>95%). When compared to free haloperidol (H-F), H-Nc showed higher therapeutic efficacy on the mania animal model, and reduced extrapyramidal side-effects (catalepsy and dyskinesia).Haloperidol is an antipsychotic drug associated with the development of movement disorders. We evaluated the effect of its nanoencapsulation on its pharmacological activity and motor side effects. Haloperidol-loaded polysorbate-coated nanocapsules (H-NC) showed nanometric size, negative zeta potential and low polydispersity indices and high encapsulation efficiency (>95%). Rats received a single dose of H-NC (0.2 mg/kg ip) and four doses of d,l-amphetamine, AMPH (8.0 mg/kg ip), injected every 3 h (0, 3, 6 and 9 h). The AMPH-induced stereotyped movements were quantified in the intervals of 15 min after each of four doses of AMPH, demonstrating greater pharmacological efficacy of the H-NC over free haloperidol (FH). The acute motor side effects were evaluated 1 h after a single dose of H-NC or its free solution (0.2 mg/kg ip). The group treated with H-NC presented lower extrapyramidal effects (catalepsy and oral dyskinesia) than those treated with FH. In the last experimental set, rats sub-chronically treated with a daily dose of H-NC (0.2 mg/kg ip) for 28 days showed a lower incidence of extrapyramidal effects than those treated with the free drug (0.2 mg/kg ip). Our findings showed the potential of using H-NC in the development of a nanomedicine aimed at increasing the efficacy of this antipsychotic drug and reducing its side effects.
Keywords: Haloperidol; Polymeric nanocapsules; Extrapyramidal effects; Motor disorders; Vacuous chewing movement; Catalepsy;